| There is increasing concern about the impacts of long-term agricultural practices on soil quality and health. Soil microorganisms are vital to the agroecosystem health through their roles in residue decomposition, nutrient cycling and their associations with other organisms. Humic substances are also extremely important attributes of soil quality. Understanding the microbial community in the soil environment has proven to be a challenging task, because of the extremely high abundance and enormous diversity of microorganisms in soils. In addition, the relationships between fertilization and the shifts of soil microbial communities are still not unanimous. The impacts of long-term fertilization on the composition and structure of humic substances also remain controversial and are still not fully understood. Globally, the challenge consists firstly in understanding how the ecosystem and particularly the soil quality/health are altered by intensive agriculture, and then, in developing new strategies that take advantage of ecological interactions within agricultural systems. While the issues are serious and diverse, common links among them could be soil microbes and soil organic matter as influenced by interactive effects of anthropogenic and natural perturbations. Strategies to address these issues involve a comprehensive characterization of microbial biomass, activities, and diversity as well as soil organic matter (SOM) since they are key indicators and component of soil quality/health. To elucidate these observed challenges, controversies and enigma, the relationships among fertilization, soil microbial properties and humic substances have been examined through the long-term Red Soil Experimental Station of the Chinese Academy of Agricultural Sciences, Hunan province, China. At this site, a wheat-corn rotation, receiving 12 different treatments was established in 1990.Microcalorimetry, plate count, PCR-DGGE and FT-IR in combination with complementary elemental and optical analyses were employed to characterize the microbial activity, biomass, diversity and humic substances as influenced by long-term fertilization in soils from Southern China. Fertilization greatly increased microbial biomass C and N (Cmic and Nmic) as well as the activities of phosphatase, urease, invertase, protease, catalase and dehydrogenase compared to the unfertilized treatment. Invertase exhibited larger variations between treatments (maximum divided by minimum=50.0), followed by dehydrogenase (30.75), urease (15.06), protease (12.48), phosphatase (5.83) and catalase (4.84). The fallow (H) and NPKM treatments showed higher enzyme activities except for catalase, whereas lower activities were recorded in the control (CK) and N treatments. The total bacterial community and the denitrifying bacteria registered higher populations in all treatments than the other microorganisms. However, generally the number of microorganisms and species were reduced in mineral fertilizers as compared with those in organic treatments. Among organic treatments, the numbers of denitrifying and aerobic nitrogen fixing bacteria enhanced more in manure alone, whereas the highest number of aerobic cellulose decomposing bacteria was observed in the fallow. Manure alone (M) enhanced the number of denitrifying and aerobic bacteria by 54.4% and 20.5%, respectively, whereas the fallow (H) increased the number of aerobic cellulose decomposing bacteria by 31.4%. The numbers of Bacteria and actinomycetes were strongly correlated with soil organic carbon (Corg) in this study, while those of cultivable fungi were largely linked with the C/N ratio. Fallow and soils amended with mineral fertilizers plus pig manure or straw increased both the DGGE band patterns and the Shannon index if compared with mineral fertilizers or with the control. The greatest similarity in the bacterial community structure occurred in the mineral treatments. However, band variations were also observed among the fertilizer treatments. Mineral treatments with lower bacterial numbers enhanced the values of the peak-time (tmax) more than did organic treatments. The peak-height (Pmax) was positively correlated (P<0.01), with soil enzymes, Cmic and Nmic, and the number of microorganisms, whereas the peak-time (tmax)was negatively connected (P<0.01) with these parameters. The microbial growth rate constant (k) was linked to bacteria (p<0.01), actinomycetes (p<0.05) and catalase (p<0.05). The total heat evolution (Q) had no relationships with any soil microbial properties (except for catalase). The values of the peak height and peak-time were opposite (positive and negative, respectively) but both parameters were substantially correlated with soil microbial properties. Higher values of total and available nutrients were found in NPKM, whereas CK showed lower contents of these nutrients. Except for Manure alone, the contents of aluminum and iron, extracted by dithionate-citrate bicarbonate (DCB) and sodium pyrophosphate (NaPP) decreased in the other treatments as compared to the control (CK). The lowest forms of aluminum and iron were recorded in the NPKS (4.65% and 0.88% for Al forms and 6.97% and 1.33% for Fe forms). The fallow (H) treatments also greatly decreased these forms as compared to the control (5.64% and 1.56% for Al forms and 8.46% and 2.34% for the Fe forms).Although, in general the FT-IR spectra did not differ noticeably among treatments, some important features were observed. Broad intense bands at 3400 and 3700 cm-1 are due to the stretching vibration of bonded and non-bonded hydroxyl groups. Small contributions of the bands at 2280 and 2360 cm-1 come from, NH2+, CO-NH2, CO-NH-, S-H, and P-H. The band at 1775 cm-1 originates from the C=O stretch of ester fatty acids. The broad band at 1630 cm-1 can be assigned to the aromatic C=C vibrations, symmetric stretching of COO- groups, and H-bonded C=O of conjugated ketones. Evidence for the presence of COOH groups was indicated by the peaks at 1120 to 1150 cm-1 which were attributed to the C-O stretches. The humin fraction was distinguished by the presence of a strong and sharp free OH stretch (3620-3700 cm-1) whereas, a strong carbonyl C=O stretch (1670-1820 cm-1) appeared in the HA fraction only. A C-N stretch (1080-1360 cm-1) was present in both the HA and FA fractions but the humin fraction lacked this type of vibration. Detailed insight on the reactivity of humic substances to fertilization was provided by calculation of O/R ratios, which were the intensities of oxygen-containing functional groups vs. aliphatic and aromatic groups. The humic acid (HA) from the NPKM and M treatments showed higher values of the O/R ratios (1.24 and 1.18, respectively) as compared to that from the 1.5 NPKM and H treatments, which significantly decreased these ratios (0.66 and 0.85, respectively). Except for NPKMR, the O/R ratios of the fulvic acid (FA) did not differ among the treatments. However, the humin (HM) from H and CK displayed the highest value of O/R ratios (about 3.23 for both). Humic substances from organic treatments showed more aliphatic nature, whereas more condensed-alkali soluble humic substances were formed in the unfertilized and mineral treatments. Among fractions, the HA formed more phenolic or aromatic structures than did the FA or HM. The mean carbon content was higher in the HM and HA fractions (48.83% and 47.96%, respectively). In addition, as compared to the control, the C, H, N and S contents of humic substances significantly increased in all treatments, whereas their oxygen content decreased. Higher values of the absorbances at 465 and 665 nm (E4/E6 ratios) were observed in the fraction of fulvic acids (with a mean of 12.32), whereas the humic acid and humin fractions displayed lower E4/E6 ratios (means of 6.03 and 5.16, respectively). For all fractions of humic substances, the lowest E4/E6 ratios were recorded in the NPKM treatment, followed by the fallow (H) and NPKS treatments as compared to the unfertilized and mineral treatments (Ck and NPK). Significant correlations of soil organic carbon with the carbon contents of HA, FA and HM (0.770,0.807 and 0.809, respectively) were also found (p<0.05) in this study.The findings demonstrated that Pmax and tmax can be used as indices of soil microbial activity, while k and Q are found to be poor indicators. The results further provided an in-dept and complementary indications about the functional and structural compositions of humic substances under long-term fertilization practices.
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